TheZebrafishResourceCore(Zf-Core)willbeanintegralcomponentoftheWashingtonUniversityinSt.Louis School of Medicine Model Organism Screening Center (wuMOSC), a multi-model organism platform for functional analysis of Undiagnosed Diseases Network (UDN) variants. It will serve as a resource for gene variantmodeling,particularlyincaseswhereanalysisisnotfeasibleininvertebratemodelorganisms,suchas D. melanogaster and/or C. elegans. We anticipate that our bioinformatic analyses will designate ~65 variants peryeartobeanalyzedinthezebrafishmodel. We will leverage the relatively short generation time, high fecundity, and external development of transparent zebrafish embryos to develop and implement a rapid, efficient, and disease variant-tailored pipelineforevaluatingthevariantpathogenicityusingCRISPR/Cas9approachesand/orRNAoverexpression. TheZf-Corehasimprovedonpreviousmethodsoffishhusbandrybyemployinglarge-scaleroboticfeedingto allow for rapid growth of animals. This effectively halves the typical zebrafish generation time and thus will accelerate screening UDN variants proposed here. The PI laboratory also recently improved homologous recombination-basedgenomeeditingmethodsinzebrafishusingTALENorCRISPR/Cas9strategies. ForUDNgene-variantswherethenullallelemanifestsaphenotypepriortoday3postfertilization,wewill assess the degree to which the mutant phenotype can be rescued by injection into one-celled zygotes of synthetic RNA encoding the human protein variant, or zebrafish protein with this variant introduced. These experimentsprovidearapidassessmentastowhetherthevarianthasnormal,reduced,elevatedorotherwise abnormal activity. If lines harboring nonsense or other deleterious mutations in the disease gene of interest already exist they will be imported to our Zf-Core. Alternatively, we will generate small insertions/deletions (indel)mutationsusingCRISPR/Cas9strategybydesigningaguideRNAthatwouldalsoallowustointroduce thediseasevariantintotheendogenouslocus. Inasecondstrategy,forgeneswithphenotypesdetectableafterday3postfertilization,thevariantwillbe knocked-into the zebrafish ortholog by CRISPR/Cas9 editing. The phenotype of the resulting loss of function andknock-inmutantswillbeanalyzedtoverifyanyreporteddefectsortouncoverthem.Transparentembryos and larvae will be evaluated at the level of overall morphology, formation of specific organs and tissues using in vivo microscopy, and adults by microCT imaging to assess skeleton and soft tissues. Observation of a phenotypewithavariantknock-insuggeststhatthevariantisdeleterioustothemodelorganismandprovides experimental evidence that it is a promising/likely disease-causing candidate. Lack of observable phenotype would indicate the experimental test provides no evidence for pathogenicity. The Zf- Core will be a strong componentofthewuMOSCtoinformthediagnosisofrareandpoorlycharacterizeddiseases.